Cis and trans RNA elements play significant roles in regulating gene expression through diverse mechanisms. Technologies designed to precisely regulate expression in response to specific cellular signals are essential tools in cellular engineering applications such as metabolic engineering, in vivo sensors, and gene therapy. This proposal uses an interdisciplinary approach spanning the areas of molecular biology and molecular engineering to develop RNA-based technologies built on flexible, modular platforms that will regulate expression levels in response to concentrations of specific cellular ligands. This research presents the first generation of 'smart' RNA regulators, called antiswitches, that will serve as powerful tools in gene therapy and targeted therapeutics by regulating the expression of specific transcripts in response to particular cellular environments that can be 'sensed' by the regulator molecule. The long-term objective of this proposal is to develop an enabling technology based on the design of synthetic, modular, RNA regulators that will directly act to regulate the expression levels of targeted genes. The specific aims under this proposal are designed to demonstrate proof of principle for the development of this platform and include: (1) generating a platform for the design of modular non-coding RNAs, or antiswitches, that act as protein-responsive RNA switches; (2) characterizing the binary response of the antiswitch platform to effector and target levels in vitro; (3) characterizing antiswitch response to endogenous biomolecules and targeting to endogenous genes; (4) expanding the platform to a cooperative regulator that requires two effector inputs to affect a targeted expression event; (5) designing an antiswitch-based signal integration scheme that functions as a gradient filter. To achieve these aims we will incorporate design and tuning strategies previously developed in our laboratory: These strategies have been applied to the design of an initial platform for a small molecule-responsive antiswitch. While emphasis will be placed on the rational design of these regulators, selection strategies will be used where appropriate. The proposal combines nucleic acid design techniques with experimental strategies for monitoring regulation of target gene expression. This work will result in a robust platform for the design of tailor-made regulators that will control the expression of target genes in response to specific effector molecules for a variety of applications. [unreadable] [unreadable] [unreadable]